Rapid and accurate differentiation of bacterial strains is important when making medical diagnoses, in epidemiological studies, and for studying evolutionary diversity among bacteria. Various methods exist for typing or detecting bacterial strains, including RFLP, hybridization, and sequencing. Epidemiologically informative microsatellite DNA polymorphisms have been observed in different strains of Helicobacter pylori (Marshall et al. (1996) J. Appl. Bacteriol. 81:509-517). Similarly, repetitive DNA elements of Mycobacterium tuberculosis have been used for efficient strain tracking (Van Soolingen et al. (1993) J. Clin. Microbiol. 31:1987-1995). In addition, short sequence repeat (SSR) variation has been used to differentiate the strains of Haemophilus influenzae isolated from different patients (van Belkum et al. (1997) Infect. Immun. 65:5017-5027). However, current methods available to specifically differentiate bacterial strains, such as Lactobacillus acidophilus strains, are based either on 16SrRNA gene sequencing, which is only accurate to the species level, or on long and difficult Pulsed Field Gel Electrophoresis (PFGE) procedures.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat; also called SPIDR (Spacers Interspersed Direct Repeats), VNTR (Variable Number of Tandem Repeats), SRVR (Short Regularly Variable Repeats), and SRSR (Short Regularly Spaced Repeats)) loci, described by Jansen et al. (2002) OMICS J. Integr. Biol. 6:23-33, constitute a novel family of repeat sequences that is present in Bacteria and Archaea but not in Eukarya. The repeat loci typically consist of repetitive stretches of nucleotides with a length of 25 to 37 base pairs alternated by nonrepetitive DNA spacers of approximately equal length. To date, CRISPR loci have been identified in more than forty microorganisms (Jansen et al. (2002) OMICS J. Integr. Biol. 6:23-33), but from the lactic acid bacteria, they have only been described from Streptococcus species. Despite their discovery over 15 years ago in E. coli (Ishino et al. (1987) J. Bacteriol. 169:5429-5433), no physiological function has yet been discovered. The nucleotide sequences of the repeats are generally highly conserved within a species, but show low similarity between species. It has also been shown that variability among CRISPR loci is not due primarily to single nucleotide base changes, but rather to deletions/insertions of entire repeat and spacer regions. These properties have led to the use of the CRISPR loci as a strain-typing tool in Mycobacterium (Groenen et al. (1993) Mol. Microbiol. 10:1057-1065).
As methods to differentiate Lactobacillus bacteria, specifically L. acidophilus, are either not accurate to the strain level or are technically demanding, the development of new methods for differentiating Lactobacillus strains is desirable.